Compositions and Methods for Identifying and Modulating Metabolic Health

ABSTRACT

This invention provides reagents, methods and biochemical markers for identifying and providing therapeutic intervention for individuals with metabolic dysfunction, or individuals at risk for metabolic dysfunction.

BACKGROUND OF THE INVENTION Field of the Invention

This disclosure provides reagents, methods and biochemical markers foridentifying and treating individuals with metabolic dysfunction, and fordetermining efficacy of a therapeutic intervention to correct metabolicdysfunction. Specifically, embodiments of the present disclosure relateto novel methods of identifying and treating metabolic disease mediatedby PLEXIN D1 (PLXND1) and COLLAGEN 5A1 (COL5A1), wherein thedistribution and morphology of adipose tissue (AT) is disrupted relativeto normal, healthy individuals.

Description of Related Art Adipose Tissue Distribution and Morphology

The regional distribution and morphology of AT are strong predictors ofmetabolic disease (Salans L B, Knittle J L, & Hirsch J (1968), TheJournal of clinical investigation 47 (1):153-165; Ahima R S & Lazar M A(2013), Science 341 (6148):856-858; Amer E & Amer P (2013), Science 342(6158):558-559.) Excess lipid deposition in visceral adipose tissue(VAT) (adipose associated with visceral organs) is associated withincreased susceptibility to insulin resistance and type 2 diabetes(Hoffstedt J, et al. (2010), Diabetologia 53 (12):2496-2503) whereasexpansion of subcutaneous adipose tissue (SAT) (adipose between muscleand skin) is associated with reduced risk of metabolic disease and iseven protective against hyperglycemia and dyslipidemia. (Id.; Fox C S,et al. (2007), Circulation 116 (1):39-48; Snijder M B, van Dam R M,Visser M, & Seidell J C (2006), International journal of epidemiology 35(1):83-92; Kim J Y, et al. (2007), The Journal of clinical investigation117 (9):2621-2637.)

In turn, hypertrophic AT morphology (few large adipocytes) is associatedwith insulin resistance and AT dysfunction; whereas hyperplastic ATmorphology (many small adipocytes) is associated with improved metabolicparameters. (Id.; Sun K, Kusminski C M, & Scherer P E (2011), TheJournal of clinical investigation 121 (6):2094-2101; Kusminski C M, etal. (2012), Nature medicine 18 (10):1539-1549.)

PLEXIN D1 Association with Metabolic Disease

Genome-wide association studies have implicated the PLEXIN D1 (PLXND1)gene as one of dozens of genes whose expression pattern are associatedwith body fat distribution and type 2 diabetes in humans. (Shungin D, etal. (2015), Nature 518 (7538):187-196.)

Prior to the present disclosure, whether or how Plxnd1 plays a direct,causal role in AT morphology, distribution, and metabolism is unknown.

Plxnd1is a transmembrane receptor that controls the migration,proliferation and survival of diverse cell types. (Gay C M, Zygmunt T, &Torres-Vazquez J (2011), Developmental biology 349 (1):1-19.) Mutationof Plxnd1 in mouse and zebrafish leads to hypervascularization in manytissues. (Gitler A D, Lu M M, & Epstein J A (2004), Developmental cell 7(1):107-116; Torres-Vazquez J, et al. (2004), Developmental cell 7(1):117-123.) Vascular endothelial cell Plxnd1 modulates extracellularmatrix (ECM) synthesis and composition by regulating the collagenreceptor, β1-Integrin. (Sakurai A, et al. (2010), Molecular and cellularbiology 30 (12):3086-3098.) In turn, ECM provides a supportivemicroenvironment for AT growth and function. (Mariman E C & Wang P(2010), Cellular and molecular life sciences: CMLS 67 (8):1277-1292.)

Type V Collagens and ECM Dynamics During Adipogenesis

Type V collagens are ECM proteins that regulate collagen fiber assembly,geometry and strength. (Wenstrup R J, et al. (2011), The Journal ofbiological chemistry 286 (23):20455-20465; Sun M, et al. (2011), Journalof cell science 124 (Pt 23):4096-4105.). In addition, type V collagensare upregulated during adipogenesis and can stimulate adipocytedifferentiation in vitro. Spencer M, et al. (2011), The Journal ofclinical endocrinology and metabolism 96 (12):E1990-1998; Nakajima I,Muroya S, Tanabe R, & Chikuni K (2002), Differentiation; research inbiological diversity 70 (2-3):84-91; Nakajima I, Muroya S, Tanabe R, &Chikuni K (2002), Biology of the cell 94 (3):197-203.)

Prior to the present disclosure, whether or how type V collagens mediatemetabolic health through a role in AT morphology, distribution, andmetabolism was unknown.

Therefore, there is a need in the art to identify factors that regulateAT distribution and morphology, and that comprise molecular targets foridentifying and treating subjects with metabolic dysfunction. Inaddition, there is a need in the art to determine whether and how thePLXND1 gene exerts control over AT distribution, morphology, andmetabolism, and by extension metabolic health. Similarly, there is aneed in the art to identify whether and how type V collagens exertcontrol over AT distribution, morphology, and metabolism, and byextension metabolic health. Still further, there is a need to developclinical methods to identify and treat metabolic diseases characterizedby abnormal or dysfunctional AT distribution and morphology.

SUMMARY OF THE INVENTION

Against this backdrop, embodiments of the present disclosure address oneor more of the above-identified needs, among others, recognized by thoseskilled in the art, and provide several benefits over existing clinicalmethods of identification and therapeutic intervention in subjects withmetabolic dysfunction.

In embodiments, the invention disclosed herein provides methods fortreating a subject (i.e. providing a “therapeutic treatment”) forsubjects, or patients, with metabolic dysfunction. In embodiments,methods of treatment are provided for subjects suffering from, orsuspected to suffer from, or with a propensity toward, metabolicdysfunction. In certain embodiments, the disclosure provides methods forpreventing metabolic dysfunction in a subject. In still furtherembodiments, the disclosure provides methods for treating or preventingmetabolic dysfunction in combination with other therapies.

In some embodiments, the disclosure provides methods of treating orpreventing metabolic dysfunction, where such metabolic dysfunction isbased on, or characterized by, changes in the ratio of Visceral AdiposeTissue (VAT) to Subcutaneous Adipose Tissue (SAT). In furtherembodiments, the disclosure provides therapeutic treatment in subjectswith metabolic dysfunction based on changes in the prevalence ofhypertrophic AT morphology (i.e., AT morphology characterized by beingcomprised of relatively few large adipocytes) and hyperplastic ATmorphology (i.e., AT morphology characterized by being comprised of manysmall adipocytes).

In still further embodiments, the disclosure provides methods oftreating or preventing metabolic dysfunction, including, but not limitedto or mutually exclusive with, diabetes mellitus type II; impairedglucose tolerance or insulin resistance; high blood pressure; centralobesity and difficulty losing weight; high cholesterol; combinedhyperlipidemia; including elevated LDL; decreased HDL; elevatedtriglycerides; and fatty liver (especially in concurrent obesity).

In embodiments, the disclosure provides methods for treating a subjectwith metabolic dysfunction, including insulin resistance and/or relatedconditions, in a subject in need thereof, the method comprising the stepof decreasing the level or activity of the Plxnd1 gene, or products ofthe Plxnd1 gene. The level of the Plxnd1 gene, or products of the Plxnd1gene, may be decreased by methods disclosed in the various embodiments.

In further embodiments, disclosure pertains to a method for preventingand/or treating metabolic dysfunction, including insulin resistanceand/or related conditions, in a subject in need thereof, the methodcomprising the step of decreasing the level or activity of one or moregenes, or their products, selected from collagen, type I, alpha 2(COL1A2 in human, col1a2 in zebrafish); collagen, type II, alpha 1(COL2A1 in human, col2a1a/col2a1b in zebrafish); collagen, type IV,alpha 1 (COL4A1 in human, col4a1 in zebrafish); collagen, type V, alpha2 (COL5A2 in human, col5a2a/col5a2b in zebrafish); collagen, type V,alpha 3 (COL5A3 in humans, col5a3a/col5a3b in zebrafish); collagen, typeVI, alpha 1 (COL6A1 in human, col6a1 in zebrafish); fibronectin 1 (FN1in human, fn1a/fn1b in zebrafish); aggrecan (ACAN in human, acana/acanbin zebrafish); laminin, alpha 1 (LAMA1 in human, lama1 in zebrafish);glypican 4 (GPC4 in human, gpc4 in zebrafish); and secreted protein,acidic, cysteine-rich (osteonectin) (SPARC in human, sparc inzebrafish). The level of the one or more genes, or products of the oneor more genes, may be decreased by methods disclosed in the variousembodiments herein.

In still further embodiments, this disclosure pertains to a method forpreventing and/or treating metabolic dysfunction, including insulinresistance and/or related conditions, in a subject in need thereof, themethod comprising the step of increasing the level or activity of theCOL5A1 gene, or products of the COL5A1 gene. The level of the COL5A1gene, or products of the COL5A1 , may be increased by methods disclosedin the various embodiments.

In embodiments, the invention disclosed herein provides methods foridentifying a subject with metabolic dysfunction. In embodiments,metabolic dysfunction identified in a subject according to the presentmethods is based on changes in the ratio of Visceral Adipose Tissue(VAT) to Subcutaneous Adipose Tissue (SAT). In some embodiments, thedisclosure further provides methods for identifying a subject withmetabolic dysfunction based on changes in the prevalence of hypertrophicAT morphology (i.e., AT morphology characterized by being comprised ofrelatively few large adipocytes) and hyperplastic AT morphology (i.e.,AT morphology characterized by being comprised of many smalladipocytes).

The disclosure also provides methods for predicting whether a subject issuffering from a metabolic dysfunction, or is at elevated risk formetabolic dysfunction. In these embodiments, the methods comprise thesteps of: (a) isolating a biosample from a subject; (b) determining alevel or concentration of one or more biomarkers present in thebiosample; and (c) identifying the subject as suffering from a metabolicdysfunction, or is at elevated risk for metabolic dysfunction, when thelevel or concentration of one or more biomarkers is increased ordecreased relative to a control level or range.

The disclosure also provides methods for identifying a subject that iseligible for reimbursement of an insurance claim for treatment ofmetabolic dysfunction. In these embodiments, the methods comprise thesteps of: (a) isolating a biosample from a subject; (b) determining alevel or concentration of one or more biomarkers present in thebiosample; and (c) as eligible for reimbursement of the insurance claimwhen the concentration of one or more biomarkers is increased ordecreased relative to an insurance control value. In these embodiments,the insurance control value refers to an amount or range of amounts of abiochemical marker such as Plxnd1 and Col5a1.

The disclosure further provides methods for determining the efficacy ofa treatment for metabolic dysfunction in a subject. In theseembodiments, the methods comprise the steps of: (a) treating a subjectfor a metabolic dysfunction; (b) isolating a biosample from the subject;(c) determining a level or concentration of one or more biomarkerspresent in the biosample; and (d) determining the efficacy of thetreatment for metabolic dysfunction when the concentration of one ormore biomarkers is increased or decreased relative to a pre-treatmentlevel or pre-treatment range of the one or more biomarkers.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description can be best understood when read inconjunction with the following drawings in which:

FIG. 1 shows that reduced VAT volume and hyperplastic morphologyunderlie an altered body fat distribution in plxnd1 mutant zebrafish.

FIG. 1A shows VAT area is reduced in plxnd1zebrafish (P=0.0115), leadingto a decreased VAT:SAT ratio (inset, P<0.0001). There were trendstowards increases in SAT (P=0.142), miscellaneous AT (P=0.053) and totaladiposity (P=0.085). FIG. 1B shows probability density functions torepresent VAT-LD diameter distributions. VAT-LD diameters were modelledusing a mixture of 2 normal distributions. The mean (μ) of eachdistribution is indicated (μ1 and μ2). FIG. 1C shows quantification ofEdU+ nuclei normalized to total nuclei from Z-stacks. FIG. 1D showsquantification of EdU+ nuclei colocalizing with lipid droplet-containingadipocytes, AT macrophages, and endothelial cells from plxnd1 and wildtype sibling variants of the fli1a:EGFP transgenic line. FIG. 1E showsthat mRNA levels for adipocyte differentiation markers cebpa, cebpb andpparg were increased in plxnd1 VAT by qRT-PCR. Fabp11a, a homolog ofmammalian Fabp4/aP2, was unchanged. FIG. 1F shows a significant positivecorrelation was observed between VAT PLXND1 mRNA and hypertrophic VATmorphology in humans.

FIG. 2 shows that Col5a1 is essential for maintenance of thehyperproliferative and hyperplastic state of plxnd1 mutant VAT.

FIG. 2A shows qRT-PCR for ECM markers from whole zebrafish VAT. AllmRNAs shown were significantly different between plxnd1 and siblings(α=0.05). FIG. 2B shows quantification of Col5 area in wild-type andplxnd1 VAT as measured by confocal immunofluorescence imaging. Area isexpressed as % of field of view. FIG. 2C shows quantification of Col5signal in endothelial cells relative to background as measured byconfocal immunofluorescence imaging. FIG. 2D shows qRT-PCR on adipocyte(Adipo.) and stromal vascular cell (SVC) fractions reveals col5a1isenriched in SVCs of plxnd1 VAT. FACS enrichment of EGFP+ endothelialcells from fli11a:EGFP plxnd1 mutant and sibling VAT show thatSVC-derived col5a1is upregulated in plxnd1 endothelial cells. FIG. 2Eshows means of probability density functions to represent VAT-LDdiameter distributions from sibling or plxnd1 animals injected witheither control or col5a1Vivo-Morpholino (vMO). VAT-LD diameters weremodelled using a mixture of 2 or 3 normal distributions. The mean (μ) ofeach distribution is indicated (μ1, μ2, μ3). FIG. 2F shows thatinjection of col5a1vMO normalizes the hyperproliferation observed inplxnd1 VAT. FIG. 2G shows that injection of col5a1vMO increases VATcumulative volume in plxnd1 mutants. FIG. 2H shows that injection ofcol5a1vMO increases the VAT:SAT ratio in plxnd1 mutants.

FIG. 3 shows that the extracellular matrix of plxnd1 mutant VAT issufficient to induce hyperplastic morphology in a Col5a1-dependentmanner.

FIG. 3A shows 3D renderings of sibling or plxnd1 mutant VAT stained withLipidTOX (lipid droplets, dark grey/black) and 5-DTAF (collagen, lightgrey/white). FIGS. 3B&C show turbidity assays using ECM extracted fromzebrafish VAT and induced polymerization and gel formation in vitro.These results reveal a Col5a1-dependent increase in (B) the rate offibrillogenesis, and (C) greater ultimate turbidity suggesting increasedcollagen fibrils within plxnd1 VAT. FIG. 3D shows a schematic of ECM andSVC 3D co-culture experimental design. Briefly, ECM and SVCs wereisolated from either sibling or plxnd1 mutant VAT. The isolated ECM wasthen used as a 3D substrate for SVC culture. FIG. 3E shows confluency (%of field occupied by cells) of adipogenic clusters after 10 days of SVC3D co-culture. SVCs were isolated from either sibling or plxnd1 VAT andused to seed either sibling or plxnd1 ECM gels (indicated on thex-axis). FIG. 3F shows mean LD size of 3D cultures for the conditionsdescribed for 3F. LD sizes were normally distributed.

FIG. 4 shows that VAT fails to expand in homozygous plxnd1 mutants fed ahigh-fat diet, leading to disproportionately large increases in SAT.

FIG. 4A shows quantitated total adipose area (Nile Red stained) relativeto standard length after 14 days of normal or high-fat diet (HFD).Groups were either plxnd1 homozygous mutants (plxnd1) or plxnd1heterozygotes (plxnd1/+). Left panels indicate whole animal body fatdistribution, with areas enlarged on the right denoted by boxes.Experimental groups are: plxnd1/+ fed control diet (top row); plxnd1/+fed high-fat diet (second row); plxnd1 fed control diet (third row);plxnd1 fed high fat diet (bottom row). Results show greater lipidstorage and deposition after HFD intervention. FIG. 4B shows plxnd1mutants fed a HFD had a greater VAT:SAT ratio indicatingdisproportionate lipid storage in SAT. FIG. 4C shows means ofprobability density functions of VAT LD sizes. All groups exhibitedbimodal LD size distributions. FIG. 4D shows means of probabilitydensity functions of SAT LD sizes. All groups exhibited unimodal LD sizedistributions.

FIG. 5 shows that mutation of plxnd1 protects zebrafish from high-fatdiet induced insulin resistance.

FIG. 5A shows basal blood glucose measurements revealing that wild-typesiblings fed a high-fat (HF) diet are hyperglycaemic compared tocontrols. FIG. 5B shows glucose tolerance tests that reveal wild-typesiblings fed a high-fat (HF) diet have a decreased capacity to normalizeblood glucose relative to control fed siblings (P<0.001). plxnd1 mutantsfed a control diet have an enhanced capacity to normalize blood glucoserelative to control fed siblings (P<0.01); whereas, plxnd1 mutants fed aHF diet have equivalent capacity to normalize experimentally-inducedhyperglycemia to control fed siblings (P>0.05). One factor ANOVAfollowed by Tukey's HSD test (α=0.05) was used to determine statisticalsignificance at 120 min. FIG. 5C shows qRT-PCR analysis of markers ofinsulin signaling and metabolism in zebrafish VAT. FIG. 5D shows humanPLXND1 mRNA is significantly upregulated in VAT, but not SAT, of obesepatients with type 2 diabetes. FIG. 5E shows schematic illustrating thecurrent working model of Plxnd1-mediated regulation of VAT morphology.

FIG. 6 shows plxnd1 zebrafish mutants have reduced lipid storage in VAT.

FIG. 6A shows Folch lipid extraction revealing similar total lipidlevels per fish between siblings and plxnd1 mutants. FIG. 6B showsquantification of the cumulative lipid droplet volume in VAT (leftpanel) or SAT (right panel). FIG. 6C shows the experimental strategyexamining the relationship between PLXND1 mRNA levels and AT morphology.FIG. 6D shows no significant correlation observed in human SAT betweenPLXND1 and hypertrophic morphology in human SAT.

FIG. 7 shows hyperplastic VAT morphology in plxnd1 zebrafish.

FIG. 7A shows comparison of μ2 VAT-LD diameter between plxnd1 andsiblings. μ2 VAT-LD diameter was significantly smaller in plxnd1. FIG.7B shows a log₂ distribution of LD volumes revealing a trimodaldistribution with plxnd1 LDs smaller than sibling LDs. FIG. 7C shows aplxnd1 mutants have increased LD number per confocal Z-stack. FIG. 7Dshows stained histological sections indicating the hyperplasticmorphology of plxnd1 VAT. Adipocytes are the large, light stainingcircular structures. The darker staining structures surroundingadipocytes are exocrine pancreatic tissue embedded within the VAT(arrows). Asterisks indicate intestinal tissue. FIG. 7E shows VATadipocyte diameter measured in histological sections. FIG. 7F showsnumber of VAT adipocytes per histological field.

FIG. 8 shows plxnd1 mutant SAT is indistinguishable from wild-typesiblings.

FIG. 8A shows maximum intensity projections of SAT LDs in plxnd1 mutantsand siblings labelled with LipidTOX. FIG. 8B shows mean SAT-LD diameterwas not significantly different between plxnd1 and siblings. FIG. 8Cshows SAT-LD number per confocal Z-stack was not different betweenplxnd1 and siblings. FIG. 8D shows the probability density function ofSAT-LD diameters modelled using a mixture of 2 normal distributions. Themean (μ) of each distribution is indicated (μ1 and μ2). Dispersion (σ)of each distribution was sibling σ1=2.51, sibling σ2=16.77, plxnd1σ1=2.57 and plxnd1 σ2=15.63. Probabilities (π) are sibling π1=0.73,sibling π2=0.27, plxnd1 π1=0.59 and plxnd1 π2=0.41.

FIG. 9 shows the experimental design and validation of col5a1 targetedVivo-Morpholino experiments.

FIG. 9A shows a schematic depicting the experimental design of col5a1vMO injection. FIG. 9B shows a schematic depicting the col5a1 vMOinjection regimens. Regimens were performed with two different col5a1vMOs. FIG. 9C shows a schematic depicting zebrafish col5a1 genestructure. vMOs were designed to target the intron 1-exon 2 (i1e2) andexon 3-intron 3 (e3i3) boundaries. Observed isoforms are schematizedbelow the gene structure. A predicted stop codon was found at theimmediate start of intron 1 for the col5a1-i1e2 isoform leading to atruncated 55 amino acid Col5a1 containing only exon 1 and a partialConcanavelin-A like lectin and Laminin G domains. For the col5a1-e3i3isoform, a premature stop codon was found at the start of exon 4producing a 112 amino acid Col5a1 containing a truncated Concanavelin-Alike lectin and Laminin G domains. Arrows indicate the location ofpremature stop codons. Locations of PCR primers used to determineisoform structure are shown and sequences are given in Table 1. Expectedproduct sizes are 601 bp for control (ctrl) col5a1 isoform, 444 bp forcol5a1-i1e2 and 224 bp for col5a1-e3i3. Isoforms were confirmed bysequencing. FIG. 9D shows RT-PCR depicting the inclusion of part ofintron 1 after injection of col5a1-i1e2, and the skipping of exon 3after injection of col5a1-e3i3. M=marker (100-1000 bp in 100 bpincrements). The induced col5a1mRNA isoforms are predicted to lead to areduction in Col5a1 function based on previous published literature.(Marchant J K, Hahn R A, Linsenmayer T F, & Birk D E (1996), The Journalof cell biology 135 (5):1415-1426.)

FIG. 10 shows Col5 reactivity is reduced after injection of col5a1 vMOs.Maximum intensity projections of immunofluorescently labelled Col5 inVAT were quantified relative to background in both endothelial cell andperi-adipocyte locations with or without col5a1 vMOs in for theindicated genotypes.

FIG. 11 shows plxnd1 mutant VAT has increased fibrous collagen asindicated by Masson's trichrome staining.

FIG. 11A shows Masson's trichrome staining (collagen, dark stain,arrows) is increased in VAT of plxnd1 mutants. FIG. 11B shows isolatedECM from plxnd1 VAT and stained with 5-DTAF exhibits increased fibrousstructures. Arrows indicate the increased diameter of plxnd1 fibers.FIG. 11C shows % collagen area. Measurements were taken on Masson'strichrome stained sections, and area is expressed as % of total field.FIG. 11D shows mean fiber diameter. Measurements were taken on maximumintensity projects of 5-DTAF stained isolated ECM.

FIG. 12 shows altered ECM composition in plxnd1 VAT.

FIG. 12A shows quantification of the area of Periodic acid Schiff (PAS)stained VAT in wild type (sib) and plxnd1 strains. FIG. 12B showsquantification of the area of Alcian blue stained VAT in wild type (sib)and plxnd1 zebrafish. FIG. 12C shows quantification of the area ofElastin stained VAT in wild type (sib) and plxnd1 zebrafish. FIG. 12Dshows quantification of collagen stained SAT (Masson's trichromehistology) in wild type (sib) and plxnd1 zebrafish.

FIG. 13 shows Isolation and characterization of ECM from zebrafish VAT.

FIG. 13A shows a schematic illustrating the process of ECM extractionfrom VAT. FIG. 13B shows a turbidity assay revealing increasedfibrillogenesis in plxnd1 mutant VAT. FIG. 13C shows maximum intensityprojections of isolated VAT ECM stained with 5-DTAF from siblings andplxnd1 reveals the increased fibrous structure of plxnd1 mutant VAT ECM.

FIG. 14 shows mixing of sibling and plxnd1 ECM induces an intermediateproliferation and morphology phenotype.

FIG. 14A shows brightfield images of representative 3D cultures after 10days of culture. FIG. 14B shows confluency of cultures after 10 days ofincubation. FIG. 14C shows mean LD size within cultures after 10 days ofincubation. FIG. 14D shows confluency of cultures after 10 days ofincubation. Cultures were from plxnd1-derived SVCs cultured with eitherplxnd1+control vMO (ctrl) or col5a1 vMO (col5a1). FIG. 14E shows mean LDsize within cultures after 10 days of incubation. Cultures were fromplxnd1-derived SVCs cultured with either plxnd1+control vMO (ctrl) orcol5a1 vMO (col5a1).

FIG. 15 shows adipose quantification in plxnd1 mutants fed a high-fatdiet.

FIG. 15A shows quantification of total adipose area in plxnd1homozygotes and plxnd1/+ heterozygotes fed either a control or high-fatdiet. FIG. 15B shows quantification of VAT area in plxnd1 homozygotesand plxnd1/+ heterozygotes fed either a control or high-fat diet. FIG.15C shows quantification of SAT area in plxnd1 homozygotes and plxnd1/+heterozygotes fed either a control or high-fat diet.

FIG. 16 shows PLXND1 mRNA levels are associated with reducedInsulin-stimulated lipogenesis in SAT in humans.

FIG. 16A shows SAT PLXND1 mRNA is inversely associated withinsulin-stimulated lipogenesis in SAT adipocytes. FIG. 16B shows SATCOL5A1 mRNA is inversely associated with insulin-stimulated lipogenesisin SAT adipocytes. FIG. 16C shows COL5A1 mRNA is significantlyupregulated in VAT, but not SAT, of obese patients with type 2 diabetes.FIG. 16D shows qRT-PCR reveals plxnd1 and col5a1 mRNAs are increasedafter feeding zebrafish a HFD for 14 days.

FIG. 17 shows a schematic model depicting VAT and SAT dynamics afterPlxnd1 manipulation.

FIG. 18 shows quantification of the ratio of lipid droplets fromzebrafish VAT categorized into small (<20 μm -60 μm diameter) and large(>60 μm diameter) lipid droplets for the indicated genotypes and vMOs.Only plxnd1 mutants injected with control vMO exhibited an increase inthe ratio of small:large LDs.

FIG. 19 shows PLXND1 mRNA levels were higher in human SAT than VAT. FIG.19A shows qRT-PCR for PLXND in human VAT and SAT. FIG. 19B shows qRT-PCRfor plxnd1 in adult zebrafish VAT and SAT.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

Before describing the disclosed methods and compositions in detail, anumber of terms will be defined. As used herein, the singular forms “a”,“an”, and “the” include plural referents unless the context clearlydictates otherwise.

It is noted that terms like “preferably,” “commonly,” and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that can or cannot be utilized in a particular embodiment ofthis invention.

For the purposes of describing and defining this invention it is notedthat the term “substantially” is utilized herein to represent theinherent degree of uncertainty that can be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation can vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

Provided herein are methods for identifying, preventing, or treating asubject at risk for metabolic dysfunction and its associated disorders.Metabolic dysfunction will be recognized in the art as including aspectrum of disorders. In embodiments of the present disclosure,conditions associated with metabolic dysfunction include, but are notlimited to or mutually exclusive with, diabetes mellitus type II (alsoreferred to as Type 2 diabetes, or T2D); impaired glucose tolerance orinsulin resistance; high blood pressure; central obesity and difficultylosing weight; high cholesterol; combined hyperlipidemia; includingelevated LDL; decreased HDL; elevated triglycerides; and fatty liver(especially in concurrent obesity).

In embodiments, the metabolic dysfunction of the present disclosure ischaracterized by abnormal distribution or morphology of adipose tissue(AT). In embodiments, the metabolic dysfunction of the presentdisclosure comprises an abnormal distribution of AT accumulating invisceral adipose tissue (VAT). In some embodiments, the metabolicdysfunction of the present disclosure comprises an abnormal distributionresulting in an elevated proportion of VAT relative to subcutaneousadipose tissue (SAT).

In still further embodiments, the metabolic dysfunction of the presentdisclosure comprises hypertrophic VAT morphology in effected subjects,relative to hyperplastic VAT morphology in subjects not suffering frommetabolic dysfunction. As used herein, “hypertrophic AT morphology”comprises adipose tissues with a reduced number of adipocytes that areof increased size, and “hypertrophic VAT morphology” comprises visceraladipose tissues with a reduced number of adipocytes that are ofincreased size. As used herein, “hyperplastic AT morphology” comprisesadipose tissues with an increased number of adipocytes that are ofdecreased size, and “hyperplastic VAT morphology” comprises visceraladipose tissues with an increased number of adipocytes that are ofreduced size.

Those skilled in the art will recognize that hypertrophic morphology andhyperplastic morphology are relative terms. Thus, hypertrophic ATmorphology in a subject suffering from metabolic dysfunction isoptionally measured relative to normal AT morphology in a healthysubject. Similarly, hyperplastic AT morphology is optionally measuredrelative to the AT morphology in a subject suffering from metabolicdysfunction.

Alternatively, hyperplastic AT morphology is measured relative to the ATmorphology of a normal subject, as, for example, wherein suchhyperplastic morphology confers a protective or prophylactic effect onthe subject.

As used herein “metabolic syndrome” refers to a patient that has acollection of indicators codified in the United States with thepublication of the National Cholesterol Education Program AdultTreatment Panel III (ATP III) guidelines in 2001. Disorders associatedwith metabolic syndrome include elevated diabetes risk, hypertension,obesity, abnormal lipid metabolism (e.g. dyslipidemia), centraladiposity, oxidative stress and its many manifestations including,stroke, ischemia, and atherosclerosis.

As used herein, the term “insulin resistance” has its common meaning inthe art. Insulin resistance is a physiological condition where thenatural hormone insulin becomes less effective at lowering blood sugars.The resulting increase in blood glucose may raise levels outside thenormal range and cause adverse health effects such as metabolicsyndrome, dyslipidemia and subsequently type 2 diabetes mellitus. Theterm “insulin resistance-related complications” and “insulinresistance-related conditions” as used herein encompass, withoutlimitation, metabolic syndrome, dyslipidemia and type 2 diabetesmellitus, as well as insulin resistance in endocrine diseases (e.g.,obese subjects with type 1 diabetes mellitus, Cushing's disease andlipodystrophy syndromes).

In embodiments, this disclosure pertains to a method for preventingand/or treating metabolic dysfunction, including insulin resistanceand/or related conditions, in a subject in need thereof, the methodcomprising the step of decreasing the level or activity of the Plxnd1gene, or products of the Plxnd1 gene. The level of the Plxnd1 gene, orproducts of the Plxnd1 gene, may be decreased by methods disclosed inthe various embodiments.

In other embodiments, this disclosure pertains to a method forpreventing and/or treating metabolic dysfunction, including insulinresistance and/or related conditions, in a subject in need thereof, themethod comprising the step of decreasing the level or activity of one ormore genes, or their products, selected from collagen, type I, alpha 2(COL1A2 in human, col1a2 in zebrafish); collagen, type II, alpha 1(COL2A1 in human, col2a1a/col2a1b in zebrafish); collagen, type IV,alpha 1 (COL4A1 in human, col4a1 in zebrafish); collagen, type V, alpha2 (COL5A2 in human, col5a2a/col5a2b in zebrafish); collagen, type V,alpha 3 (COL5A3 in humans, col5a3a/col5a3b in zebrafish); collagen, typeVI, alpha 1 (COL6A1 in human, col6a1 in zebrafish); fibronectin 1 (FN1in human, fn1a/fn1b in zebrafish); aggrecan (ACAN in human, acana/acanbin zebrafish); laminin, alpha 1 (LAMA1 in human, lama1 in zebrafish);glypican 4 (GPC4 in human, gpc4 in zebrafish); and secreted protein,acidic, cysteine-rich (osteonectin) (SPARC in human, sparc inzebrafish). The level of the one or more genes, or products of the oneor more genes, may be decreased by methods disclosed in the variousembodiments herein.

In still further embodiments, this disclosure pertains to a method forpreventing and/or treating metabolic dysfunction, including insulinresistance and/or related conditions, in a subject in need thereof, themethod comprising the step of increasing the level or activity of theCol5a1 gene, or products of the Col5a1 gene. The level of the Col5a1gene, or products of the Col5a1, may be increased by methods disclosedin the various embodiments.

In yet additional embodiments, this disclosure pertains to a method forpreventing and/or treating metabolic dysfunction, including insulinresistance and/or related conditions, in a subject in need thereof, themethod comprising the step of increasing or decreasing the level oractivity of components in the intracellular pathway mediated by Plxnd1signaling. In some embodiments, the methods of the present disclosurecomprise modulating the level or activity of components upstream ofPlxnd1 signaling. In embodiments, modulating the level or activity ofcomponents upstream of Plxnd1 signaling comprises modulating the genes,or gene products, of, without limitation, members of the Neuropilin orSemaphorin family of proteins.

As used herein, the term active pharmaceutical ingredient (API) means acompound or compounds with the ability to modulate metabolic dysfunctionin a subject. In some embodiments, an API of present disclosure iscapable of modulating the levels or activity of the Plxnd1 gene, or ofproducts of the Plxnd1 gene. In other embodiments, an API of presentdisclosure is capable of modulating the levels or activity of the Col5a1gene, or of products of the Col5a1 gene. In certain embodiments, an APIof the present disclosure is capable of modulating the Plxnd1/Col5a1pathway. In still further embodiments, an API according to the presentdisclosure comprises a molecule that regulates ECM components invisceral adipose tissues.

In some embodiments, an API of the present disclosure comprises aninterfering molecule. As used herein, the term “interfering molecule”refers to any molecule that is capable of disrupting, or inhibiting, anintracellular signaling pathway. In preferred embodiments, theinterfering molecule is capable of disrupting the signaling pathway. Aninterfering molecule of the invention, for example, can inhibit theactivity of a protein that is encoded by a gene either directly orindirectly. Direct inhibition can be accomplished, for example, bybinding to a protein and thereby preventing the protein from binding anintended target, such as a receptor. Indirect inhibition can beaccomplished, for example, by binding to a protein's intended target,such as a receptor or binding partner, thereby blocking or reducingactivity of the protein.

Furthermore, an interfering molecule of the invention can inhibit a geneby reducing or inhibiting expression of the gene, inter alia byinterfering with gene expression (transcription, processing,translation, post-translational modification), for example, byinterfering with the gene's mRNA and blocking translation of the geneproduct or by post-translational modification of a gene product, or bycausing changes in intracellular localization.

Examples of suitable interfering molecules include, but are not limitedto, small molecules, antibodies, antisense RNAs, cDNAs,dominant-negative forms of molecules such as, without limitation,Plxnd1, Neuropilin, or Semaphorin peptides, protein kinase inhibitors,combinations thereof, and the like.

In still other embodiments, an API according to the present disclosurecomprises an agonist. As used herein, the term “agonist” is used in thebroadest sense and includes any molecule that mimics a biologicalactivity of a native polypeptide disclosed herein. An agonist can be anychemical compound, nucleic acid molecule, peptide or polypeptide canenhance activity of a gene product (e.g., by stabilizing the geneproduct, preventing its proteolytic degradation or increasing itsenzymatic or binding activity or directly activating expression of agene).

An agonist of the invention can increase the activity of a protein thatis encoded by a gene either directly or indirectly. Direct activationcan be accomplished, for example, by binding to a protein and therebyenhancing binding of the protein to an intended target, such as areceptor. Indirect activation can be accomplished, for example, bybinding to a protein's intended target, such as a receptor or bindingpartner, and enhancing activity, e.g. by increasing the effectiveconcentration of the target. Furthermore, an agonist of the inventioncan activate a gene by increasing expression of the gene, e.g., byincreasing gene expression (transcription, processing, translation,post-translational modification), for example, by stabilizing the gene'smRNA or blocking degradation of the mRNA transcript, or bypost-translational modification of a gene product, or by causing changesin intracellular localization.

Suitable agonist molecules specifically include agonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, small organic molecules, etc. Methods foridentifying agonists of a native polypeptide may comprise contacting anative polypeptide with a candidate agonist molecule and measuring adetectable change in one or more biological activities normallyassociated with the native polypeptide.

In some embodiments, an agonist of the present disclosure comprises amolecule capable of activating components downstream in the Plxnd1signaling pathway. In some embodiments, an agonist of the presentdisclosure is capable of activating or increasing levels or activity ofthe Col5a1 gene, or of products of the Col5a1 gene. In otherembodiments, an agonist of the present disclosure is capable ofactivating components of the Plxnd1 pathway that are negativelyregulated by the Plxnd1 gene product.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a pharmaceutical ingredient refers to a nontoxic butsufficient amount of the ingredient to provide the desired effect. Forexample one desired effect would be the prevention or treatment ofinsulin resistance, hypoglycemia, hyperglycemia, or other forms ofmetabolic dysfunction, as measured, for example, by an increase in bloodglucose level. An alternative desired effect for the peptides of thepresent disclosure would include treating hyperglycemia, e.g., asmeasured by a change in blood glucose level closer to normal, orinducing weight loss/preventing weight gain, e.g., as measured byreduction in body weight, or preventing or reducing an increase in bodyweight, or normalizing body fat distribution.

In embodiments, an effective or therapeutically effective amount iscapable of reducing the level of VAT in a subject. In some embodiments,an effective or therapeutically effective amount is capable of reducingthe VAT:SAT ratio in a subject. In other embodiments, an effective ortherapeutically effective amount is capable of reducing the level orproportion of hypertrophic VAT morphology in a subject. In still otherembodiments, an effective or therapeutically effective amount is capableof increasing the level or proportion of hyperplastic VAT morphology ina subject.

The amount that is “effective” will vary from subject to subject,depending on the age and general condition of the individual, mode ofadministration, and the like. Thus, it is not always possible to specifyan exact “effective amount.” However, an appropriate “effective” amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

As used herein, the term “patient” or “subject” refers to mammals,including humans, animal pets, farm animals, zoo animals, and the like.Further, the patient or subject of the present disclosure may refer toany vertebrate species. In one embodiment, the patient or subject is ahuman.

As used herein, the terms “treating” or “treatment” refer to theadministration of one or more APIs to a patient who has a condition ordisorder or a predisposition toward a condition or disorder, with thepurpose to alleviate, relieve, remedy, ameliorate, improve, slow or stopthe progression or worsening of the disease, or at least one symptom ofthe disease, condition or disorder, or the predisposition toward thecondition or disorder. Thus, “treating” includes prophylaxis of thespecific disorder or condition, or alleviation of the symptomsassociated with a specific disorder or condition and/or preventing oreliminating said symptoms. For example, as used herein the term“treating diabetes” will refer in general to altering glucose bloodlevels in the direction of normal levels and may include increasing ordecreasing blood glucose levels depending on a given situation.

Methods well known to those skilled in the art can be used to practiceembodiments of the present disclosure. See, for example, techniques asdescribed in Maniatis et al., 1989, MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, New York; Sambrook, J. et al., 2001, “MOLECULARCLONING: A LABORATORY MANUAL,” 3.sup.rd edition, Cold Spring HarborLaboratory Press. The contents of the above are incorporated in theirentirety herein by reference.

Additional methods well known to those skilled in the art can be used toprepare pharmaceutically acceptable compositions and methods oftreatment according to the present disclosure. See, for example, Goodman& Gilman, 2005, “THE PHARMACOLOGICAL BASIS OF THERAPEUTICS,” 11.sup.thEdition, McGraw-Hill. The contents of the above are incorporated intheir entirety herein by reference.

Additional methods well known to those skilled in the art can be usedfor therapeutic intervention in subjects with metabolic dysfunction.(See e.g. Physician's Desk Reference, Medical Economics Company, Inc.Montvale, N.J. (54th Edition) 2000; American Association of ClinicalEndocrinologists Medical Guidelines for Clinical Practice for GrowthHormone use in Adults and Children-2003 Update, AACE Growth Hormone TaskForce, Endocrine Practice (2003), 9:65-76.)

The disclosure also provides methods for predicting whether a subject issuffering from a metabolic dysfunction, or is at elevated risk formetabolic dysfunction. In these embodiments, the methods comprise thesteps of: (a) isolating a biosample from a subject; (b) determining alevel or concentration of one or more biomarkers present in thebiosample; and (c) identifying the subject as suffering from a metabolicdysfunction, or is at elevated risk for metabolic dysfunction, when thelevel or concentration of one or more biomarkers is increased ordecreased relative to a control level or range.

The disclosure also provides methods for identifying a subject that iseligible for reimbursement of an insurance claim for treatment ofmetabolic dysfunction. In these embodiments, the methods comprise thesteps of: (a) isolating a biosample from a subject; (b) determining alevel or concentration of one or more biomarkers present in thebiosample; and (c) as eligible for reimbursement of the insurance claimwhen the concentration of one or more biomarkers is increased ordecreased relative to an insurance control value. In these embodiments,the insurance control value refers to an amount or range of amounts of abiochemical marker.

The insurance control value refers to an amount or range of amounts ofone or more biochemical markers found in a comparable biosample insubjects not suffering from metabolic dysfunction, and used as aninsurance reimbursement criterion by, inter alia, a health insurer. Inanother embodiment, insurance coverage of an individual is assessed as afunction of actuarial data that is obtained from individuals withchanges in concentration of the one or more biomarkers disclosed herein.A control level according to embodiments of the present methods is basedon a database of biochemical marker such comprising one or morebiomarkers from previously tested subjects who did not exhibit ordevelop metabolic dysfunction over a clinically relevant time frame.Additionally, a control level according to embodiments of the presentmethods is based on an individual that did not file a reimbursementclaim based on metabolic dysfunction within an actuarially relevant timeperiod.

The disclosure also provides methods for determining the efficacy of atreatment for metabolic dysfunction in a subject. In these embodiments,the methods comprise the steps of: (a) treating a subject for ametabolic dysfunction; (b) isolating a biosample from the subject; (c)determining a level or concentration of one or more biomarkers presentin the biosample; and (d) determining the efficacy of the treatment formetabolic dysfunction when the concentration of one or more biomarkersis increased or decreased relative to a pre-treatment level orpre-treatment range of the one or more biomarkers.

As used herein “pre-treatment level” or “pre-treatment range” refers toa level or concentration of one or more biomarkers in a biosampleisolated from a subject before administering treatment for a disordercharacterized by metabolic dysfunction. A pre-treatment level orpre-treatment range includes, without limitation, an average of multiplemeasurements of the level or concentration of one or more biomarkers, orrange of one or more biomarkers, based on multiple measurements from asubject.

In some embodiments of the present disclosure, the level orconcentration of one or more biomarkers is increased relative to acontrol level or range. In other embodiments, the level or concentrationof one or more biomarkers is decreased relative to a control level orrange. In still other embodiments, the level or concentration of one ormore biomarkers is decreased, whereas the level or concentration ofother biomarkers are increased, relative to a control level or range.

In embodiments, the level or concentration of one or more biomarkerschanges by at least about 10 percent, for example, by at least about 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 percent,relative to a control level or range. In some embodiments, the level orconcentration of one or more biomarkers changes by at least about2-fold, for example, at least about 4, 6, 8, 10, 20, 40, 60, 80, or 100fold, relative to a control level or range.

As used herein, the term “biological sample” or “biosample” or “sample”isolated from a subject includes, but is not limited to, a tissue orbodily fluid obtained from an animal, preferably a mammal and mostpreferably a human. For example, a biological sample can be biopsymaterial, bone marrow samples, blood, blood plasma, serum or cellularfraction thereof, urine, feces, saliva, tears, or cells derived from abiological source. In one embodiment, the mammal is a human suspected ofhaving or previously diagnosed as having or in need of screening formetabolic dysfunction, in particular insulin resistance or diabetes. Incertain embodiments, a biological sample is a sample of adipose tissue.

As used herein “concentration” refers to both percent concentration andabsolute concentration of a biomarker. “Percent concentration” refers tothe comparative concentration of a biomarker with respect to another.“Absolute concentration” refers to a direct measurement of the biomarkerwithout comparison to other detected species.

A “control level” as used herein refers to an amount or range of amountsof a biochemical marker, such as, without limitation, Plxnd1 or Col5a1,found in a comparable biosample in subjects not suffering from metabolicdysfunction, metabolic syndrome or Type II diabetes. The control levelcan also be based on a database of biochemical markers such as frompreviously tested subjects who did not convert to metabolic dysfunction,metabolic syndrome or diabetes over a clinically relevant time.

In embodiments, the one or more biomarkers comprise the genes, or geneproducts, selected from collagen, type I, alpha 2 (COL1A2 in human,col1a2 in zebrafish); collagen, type II, alpha 1 (COL2A1 in human,col2a1a/col2a1b in zebrafish); collagen, type IV, alpha 1 (COL4A1 inhuman, col4a1 in zebrafish); collagen, type V, alpha 2 (COL5A2 in human,col5a2a/col5a2b in zebrafish); collagen, type V, alpha 3 (COL5A3 inhumans, col5a3a/col5a3b in zebrafish); collagen, type VI, alpha 1(COL6A1 in human, col6a1 in zebrafish); fibronectin 1 (FN1 in human,fn1a/fn1b in zebrafish); aggrecan (ACAN in human, acana/acanb inzebrafish); laminin, alpha 1 (LAMA1 in human, lama1 in zebrafish);glypican 4 (GPC4 in human, gpc4 in zebrafish); and secreted protein,acidic, cysteine-rich (osteonectin) (SPARC in human, sparc inzebrafish). In some embodiments, the control sample is a biologicalsample from a normal subject, i.e. an individual with normal metabolicfunction, or one who responds to therapy for a condition characterizedby metabolic dysfunction. In a particular aspect, the biological sampleis comprised of adipose tissue.

In some embodiments, a panel of biomarkers capable of predicting theoccurrence of metabolic dysfunction, determining the efficacy of atreatment for metabolic dysfunction is provided. Embodiments of abiomarker panel are comprised of two or more biomarkers. In oneembodiment, a biomarker panel comprises the genes, or gene products ofPLXND1/plxnd1 and COL5A1 col5a1.

In other embodiments, the subject is then included or enrolled in aninsurance plan based on the insurable status of the subject or whereinthe rate or cost of the insurance is based on the insurable status ofthe subject. Alternatively, the subject is then excluded from aninsurance plan based on the insurable status of the subject. In somesuch instances, an organization that provides medical insurance requestsor otherwise obtains information concerning a subject's biochemicalmarker status and uses that information to determine an appropriatemedical insurance premium or reimbursement of an insurance claimrelating to treatment of the subject.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

The Examples that follow are illustrative of specific embodiments of theinvention and various uses thereof. They are set forth for explanatorypurposes only and are not to be taken as limiting the invention.

EXAMPLES Example 1 Methods of Zebrafish plxnd1 Engineering and Analysis

Zebrafish husbandry and staging. To test the role of Plxnd1 in body fatdistribution, Zebrafish were genetically engineered to disrupt thefunction of the Plxnd1 gene according to methods known to the art,described in the present disclosure, as well as methods and referencesincorporated herein. Zebrafish were raised, fed, and housed asdescribed. (Westerfield M (2000) The Zebrafish Book-A guide for thelaboratory use of zebrafish (Danio rerio). University of Oregon Press,Eugene, Oreg.; Flynn E J, et al. (2009), Journal of lipid research 50(8):1641-1652; Imrie D & Sadler K C (2010), Developmental dynamics. 239(11):3013-3023.) The plxnd1^(fov01b) null mutation was maintained ontransgenic Tg(fli1a:egfp)^(y1) (hereafter referred to as fli1a:EGFP) orTg(kdrl:HsHRAS-mCherry)^(s896) (hereafter referred to as flk1:mcherry)on Ekkwill backgrounds. (Torres-Vazquez J, et al. (2004), Developmentalcell 7 (1):117-123; Childs S, et al. (2002), Development 129(4):973-982; Chi NC, et al. (2008), Genes Dev 22 (6):734-739; Jin S W,et al. (2005), Development 132 (23):5199-5209; Lawson N D & Weinstein BM (2002), Developmental biology 248 (2):307-318. VAT surrounding thepancreas and SAT at the horizontal myoseptum were used as representativeVAT and SATs. (Imrie D & Sadler K C (2010), Developmental dynamics. 239(11):3013-3023.) Experiments were conducted on zebrafish between 30-50days post fertilization, unless otherwise stated, and standard length(SL) was used to stage zebrafish. (Parichy D M, et al. (2009),Developmental dynamics 238 (12):2975-3015.)

Zebrafish lipid staining, imaging and morphometrics. Nile Red (Sigma,#N1142) and LipidTOX (Invitrogen, #H34476) staining were undertaken asdescribed. (Minchin J E & Rawls J F (2011), Methods in cell biology105C:63-86.) Live Nile Red stained animals were imaged on a Leica M205stereomicroscope. For confocal analyses, zebrafish were euthanized in1.34 g/L MS222, AT dissected and fixed in 4% paraformaldehyde overnightat 4 C before staining with LipidTOX. Hoechst and 5-Ethynyl Uridine(EdU) were used from the Click-iT EdU Imaging Kit (Invitrogen, #C10338).5-DTAF (Anaspec, #81001) was used at 200 μg/ml in 0.1M NaHCO₃ for 2 h.(Lackey D E, et al. (2014), American journal of physiology.Endocrinology and metabolism 306 (3):E233-246.) Specimens were mountedas described (Minchin J E & Rawls J F (2011), Methods in cell biology105C:63-86.). Z-stacks were obtained either on (i) an Olympus FV1000MPEmultiphoton confocal microscope equipped with a 20×1NA water dippingobjective, or (ii) a Zeiss 780LSM equipped with a 20×1NA water dippingobjective. All operations on Z-stacks were conducted in Fiji/ImageJversion 1.47n. Z-stacks were deconvolved and processed beforesegmentation. LDs were segmented using the fast marching method anddistance weighted interpolation as implemented within TrakEM2. (CardonaA, et al. (2010), PLoS biology 8 (10); Cardona A, et al. (2012), PloSone 7 (6):e38011. LD volume and Feret's diameter were quantified usingthe 3D suite. (Iannuccelli E, et al. (2010), Bioinformatics 26(5):696-697.) Analyses were performed by multiple operators with noknowledge of specimen genotype.

Zebrafish immunofluorescence and histological staining.Immunohistochemistry was performed as previously described (Kanther M,et al. (2011), Gastroenterology 141 (1):197-207.) Immunohistochemistrywas conducted with antibodies to Type V collagen (Co15) (Rockland,#600-401-107S), Laminin (Sigma, #L-9393), WCL15 (Romano N, et al.(1998), Anatomy and embryology 198 (1):31-41; van der Sar AM, et al.(2004), Trends in microbiology 12 (10):451-457), and Alexa 568(Invitrogen, #A-11011). Transmission electron microscopy was carried outas described previously. (Flynn E J, et al. (2009), Journal of lipidresearch 50 (8):1641-1652.) Whole 10-12 mm SL fish were processed forparaffin sectioning and Masson's trichrome, combined Masson's andElastin, periodic acid Schiff (PAS) and Alcian Blue stains.(Sabaliauskas N A, et al. (2006), Methods 39 (3):246-254.)

Zebrafish qRT-PCR. RNA extraction, PCR and quantitative RT-PCR (qRT-PCR)was performed as previously described using total RNA from dissectedwhole VAT tissue of 10 mm zebrafish. (Rawls J F, et al. (2007), PNAS 104(18):7622-7627.) VAT and SAT were dissected from adult Ekkwill strainwild-type zebrafish and used to assess plxnd1 mRNA levels (FIG. S18B).Samples contained 5-8 siblings or mutants per group, and qRT-PCR was ranwith three biological replicate groups, and reactions ran in triplicate.Primer sequences are listed in Table 1.

TABLE 1 Gene Forward (5′-3′) Reverse (5′-3′) symbol Gene name(SEQ ID NO.) (SEQ ID NO.) pck1 Phosphoenolpyruvate CATCACGCATCGCTAAGCTCTCAGATTCCCTT carboxykinase 1 AGAG CTTTGTC (SEQ ID NO.: 01)(SEQ ID NO.: 02) irs1 insulin receptor GAGAGCAACATGTTCC TCTGAGGTCCGGCTTGsubstrate 1 TGATTGGAATGCT CAGTGAATTGG (SEQ ID NO.: 03) (SEQ ID NO.: 04)irs2 insulin receptor GGCTTTAAGACTGGCG TTAAGATGAGGTGCAA substrate 2GTTGTTGTAA AGGTCACGG (SEQ ID NO.: 05) (SEQ ID NO.: 06) ptpn6protein tyrosine ATATTCAGAGCAGAGT GGGTGCAGATGAGCGC phosphatase, non-AAATCAG AGTTC receptor type 6 (SEQ ID NO.: 07) (SEQ ID NO.: 08) insrainsulin receptor a CAACATGCCCCCTCAC CGACACACATGTTGTT CACT GTG(SEQ ID NO.: 09) (SEQ ID NO.: 10) insrb insulin receptor bGACTGATTACTATCGC TCCAGGTATCCTCCGT AAGGG CCAT (SEQ ID NO.: 11)(SEQ ID NO.: 12) plxnd1 Plexin d1 AGAACCCCAAACTGAT ATCTGCTGTTTGATGG GCTGCACA (SEQ ID NO.: 13) (SEQ ID NO.: 14) 18S CACTTGTCCCTCTAAGTTGGTTGATTCCGATA AAGGCA ACGAACGA (SEQ ID NO.: 15) (SEQ ID NO.: 16)fabp11a Fatty acid binding GGCAAACTTGTGCAGA GAACTGAGCCTGGCAT protein 11aAACA CTTC (SEQ ID NO.: 17) (SEQ ID NO.: 18) cebpa CCAAT/enhancerATCAGCGCCTACATTG TTGCTTGGCTGTCGTA binding protein ATCC GATG(C/EBP), alpha (SEQ ID NO.: 19) (SEQ ID NO.: 20) cebpb CCAAT/enhancerCTGAGGGGAACAAGAG AGTCTGGTACGGCAGG binding protein CAAG TACG(C/EBP), beta (SEQ ID NO.: 21) (SEQ ID NO.: 22) pparg peroxisomeTGCCGCATACACAAGA ATGTGGTTCACGTCAC proliferator- AGAG TGGAactivated receptor (SEQ ID NO.: 23) (SEQ ID NO.: 24) gamma col1a2collagen, type I, ACCAGGCAGTCCAGAA GGTTTCCATTCTCAGC alpha 2 CATC ATCC(SEQ ID NO.: 25) (SEQ ID NO.: 26) col2a1a collagen, type II,GAACTTCCTCAGGCTG TGTAAGCCACGCTGTT alpha 1a CTGT CTTG (SEQ ID NO.: 27)(SEQ ID NO.: 28) col4a1 collagen, type IV, CAGGAAGGCCAGGACTCGTTCACCTGGAAATC alpha 1 ACAA CTCT (SEQ ID NO.: 29) (SEQ ID NO.: 30)col5a1 procollagen, type V, CACCCTATGCCTTATC TGTTTCATTTGCTCAA alpha 1AGTCTTC TCTCCA (SEQ ID NO.: 31) (SEQ ID NO.: 32) col5a2acollagen, type V, TACACGTGGTCAAGGA TCCCCTCACACCAGTA alpha 2a A GGTC (SEQ ID NO.: 33) (SEQ ID NO.: 34) col5a3a collagen, type V,AGGGTAAACATGGTCC ACCGATTGCACCACTT alpha 3a AGCA TCTC (SEQ ID NO.: 35)(SEQ ID NO.: 36) col5a3b collagen, type V, GATTACTGCCACACCCTCCTCAAACTCCTCCT alpha 3b ACATTC CCACA (SEQ ID NO.: 37) (SEQ ID NO.: 38)col6a1 collagen, type VI, GATGTGTGCTGCTCCT GCCCCAAAGTCTCCTT alpha 1 TTGATTTC (SEQ ID NO.: 39) (SEQ ID NO.: 40) fn1 fibronectin 1aGGAGGGATCCTGTCTG TTGCTACCTTGAGCCT ACTG TGCT (SEQ ID NO.: 41)(SEQ ID NO.: 42) acana aggrecan a GACCAAACCAGCCTGA TGCATGTAAAAGGCAG CAATATGG (SEQ ID NO.: 43) (SEQ ID NO.: 44) lama1 laminin, alpha 1ATGCTTCCGCAGATCT ACCGTCATGAGCTCGT TCAT CTCT (SEQ ID NO.: 45)(SEQ ID NO.: 46) gpc4 glypican 4 GCATGTTTCGACTGGT CCTGCTGACACACTCC CAACATGT (SEQ ID NO.: 47) (SEQ ID NO.: 48) sparc secreted protein,AAGAGGAGCCAGCTGT ATGGTTTAGGCAGGGG acidic, cysteine-rich TGAA TTCT(osteonectin) (SEQ ID NO.: 49) (SEQ ID NO.: 50) col5a1procollagen, type V, TTTCCCAGAGATGGGT AGGAACGACTGACTGC alpha 1 TGTG CTTT(SEQ ID NO.: 51) (SEQ ID NO.: 52) col5a1 procollagen, type V,CAGACGGTGTAACGAA GGGTGCAGAAACCTCA alpha 1 ACTACAG CAGT (SEQ ID NO.: 53)(SEQ ID NO.: 54)

Zebrafish plxnd1 mutants have reduced lipid accumulation in VAT andaltered body fat distribution.

Homozygous plxnd1 null zebrafish mutants and their phenotypically normalsiblings were stained with the neutral lipid dye Nile Red, andindividual ATs were categorized into; (i) VAT, (ii) SAT or (iii)miscellaneous AT (cranial or associated with the skeleton). Total ATarea, and the AT area for each category, was then measured (AT area isknown to accurately predict triacylglyceride content in zebrafish).(Tingaud-Sequeira A, et al. (2011), Journal of lipid research 52(9):1765-1772.)

Total AT area per zebrafish was indistinguishable between plxnd1 mutantsand siblings (FIG. 1A), and total extracted lipid levels (Folch, J., M.Lees, and G. H. Sloane Stanley (1957) Journal of biological chemistry226: 497-509.) per fish were identical (FIG. 6A). However, VAT area andvolume were significantly decreased in plxnd1 mutants (FIG. 1A, FIG.6B). By contrast, no significant change was observed between plxnd1mutants and siblings in SAT or miscellaneous AT-localized lipid storage(FIG. 1A, FIG. 6B). The decrease in VAT area in plxnd1 mutants led to areduced VAT:SAT ratio (FIG. 1A).

Plxnd1 deficiency induces a hyperproliferative and hyperplastic state inzebrafish VAT

LD number and size were quantified as measures of hyperplastic andhypertrophic AT morphology. (McMenamin S K, et al. (2013), Endocrinology154 (4):1476-1487.) Both sibling and plxnd1 mutant VAT had a bimodaldistribution of LD sizes containing a population of very small LDs thatwas unaltered between genotypes (FIG. 1D), and a second population oflarge LDs that was significantly smaller in plxnd1 mutants compared tosiblings (FIG. 1D and FIG. 7A). Measurements of LD volume supported thesmaller size of plxnd1 mutant VAT LDs (FIG. 7B). Furthermore, plxnd1mutants had a greater number of LDs per unit volume (FIG. 7C), andhistology confirmed the hyperplastic morphology of plxnd1 mutant VAT(FIG. 7 D-F).

plxnd1 mutant VAT had a greater number of EdU⁺ proliferating cells thansibling VAT (FIG. 1C), of which the majority of EdU⁺ nuclei belonged toeither adipocytes or endothelial cells (FIG. 1D). Further, qRT-PCRrevealed increased expression of adipocyte differentiation genes (FIG.1E). By contrast, the morphology of plxnd1 SAT was indistinguishablefrom siblings (FIG. 8). Together, these data demonstrate that Plxnd1deficiency induces adipocyte hyperproliferation, induction of adipocytedifferentiation genes and hyperplastic VAT morphology, without affectingSAT morphology.

Example 2 PLXND1mRNA is Positively Associated with HypertrophicMorphology in Human VAT, but not SAT

Human gene expression and morphology methods. Subjects were investigatedin the morning after an overnight fast. To relate PLXND1 expression toAT morphology (FIG. 1F), VAT and SAT biopsies were taken from 79 obesewomen scheduled for gastric bypass surgery. Morphology in each adiposedepot (hyperplastic/hypertrophic) was determined as described.(Hoffstedt J, et al. (2010), Diabetologia 53 (12):2496-2503.) Neithermorphology, nor hypertrophic AT were dependent on BMI. mRNA wasquantified in AT as described. (Ryden M, et al. (2007), American journalof physiology. Endocrinology and metabolism 292 (6):E1847-1855.) qRT-PCRwas performed using pre-TaqMan kits for PLXND1 (HS 00391129_m1), andLRP10 (Hs01047362_m1) (Applied Biosystems). Expression of PLXND1 wasnormalized to the LRP10 internal control using the comparative C_(t)method. PLXND1 mRNA expression was found to be slightly higher in SATthan VAT (FIG. 19A).

Multiple regression analysis (adjusting for age and body mass index)revealed a positive association between VAT PLXND1 mRNA and morepronounced hypertrophic morphology in VAT (R²=0.22 and P=0.031) inhumans (FIG. 1F). Whereas, no correlation was observed between PLXND1mRNA and morphology in SAT (FIG. 6D).

Example 3 ECM Dynamics in PLXND1Mutant Zebrafish

Col5a1 is induced by vascular endothelial cells of plxnd1 mutantzebrafish VAT

qRT-PCR revealed large-scale dysregulation of ECM components withinplxnd1 mutant VAT (FIG. 2A; see also Table 1 for qRT-PCR primers). Asubset of ECM components were downregulated (FIG. 2A); however, the typeV collagens col5a1and col5a3b were increased (FIG. 2A).

Immunofluorescence confirms an increase in Col5 protein and revealsspecific localization to vascular endothelial cells in plxnd1 mutant VAT(FIGS. 2B & C). FACS enrichment of endothelial cells followed by qRT-PCRshows that col5a1 mRNA is increased in plxnd1 mutant endothelial cells(FIG. 2D), supporting a vascular endothelial cell origin for Col5a1 inplxnd1 mutant VAT.

Knockdown of Col5a1 normalizes hyperproliferation and hyperplasticmorphology within plxnd1 mutant VAT

Zebrafish col5a1 was targeted with multiple, non-overlappingVivo-Morpholinos (vMOs) (FIG. 9).

Zebrafish were raised under normal conditions until 30-50 days postfertilization. Fish were anesthetized in 0.67 g/L MS222 and SL measuredusing an eyepiece reticle. Intra-abdominal injections were thenperformed on a Nanoject II injector (Drummond) as previously described.(McMenamin S K, et al. (2013), Endocrinology 154 (4):1476-1487.) At eachinjection, dosage was adjusted according to body weight (mg) using thefollowing equation: mg=−48.10+7.36*SL+0.68*(SL−8.94)². (McMenamin S K,et al. (2013), Endocrinology 154 (4):1476-1487.) Concentration ofindividual compounds at injection were (relative to volume/mass offish): 80 ng/mg col5a1-i1e2 vMO (5′-GAAACATGGATGCTACAGAGAGAGA-3′; SEQ IDNO.: 55) or col5a1-e3i3 vMO (5′-GAGTTCCTACTTACCTCAAACACCT-3′; SEQ IDNO.: 56) (Gene-Tools, LLC), 100 μm EDHB (Sigma) and 80 ng/mg EdU.Control animals were injected with either a standard control vMO(5′-CCTCTTACCTCAGTTACAATTTATA-3′; SEQ ID NO.: 57) (Gene-Tools) or 0.1%DMSO.

Serial injection of either vMO disrupted splicing of col5a1, and RT-PCRfollowed by sequencing confirmed the production of truncated col5a1mRNAs predicted to be non-functional (col5a1-i1e2, 55 amino acid;col5a1-e3i3, 112 amino acid) (FIGS. 9C & D). Assessment of Col5reactivity after injection of col5a1-i1e2 vMO revealed significantlyreduced Col5 protein levels in both endothelial cell and peri-adipocytelocations (FIG. 10). Injection of either col5a1vMO did not affectproliferation or morphology of sibling VAT (FIG. 2E & F). However, inhyperplastic plxnd1 mutant VAT, injection of col5a1 vMO increased LDhypertrophy and induced the appearance of an additional population ofvery large LDs (μ3=122.59 μm; FIG. 2D). Further, col5a1 vMO normalizedlevels of EdU⁺ nuclei in plxnd1 mutant VAT (FIG. 2F), and volumetricanalysis revealed a partial rescue of lipid storage in plxnd1 mutant VAT(FIG. 2G), and increasing VAT:SAT ratio (FIG. 2H).

plxnd1 mutant VAT undergoes augmented fibrillogenesis in aCol5a1-dependent manner

The fluorescent collagen probe 5-(4,6-Dichlorotriazinyl)Aminofluorescein (5-DTAF) labels VAT-localized collagen fibers to revealthe architectural properties of ECM. (Lackey D E, et al. (2014),American journal of physiology. Endocrinology and metabolism 306(3):E233-246.) The ECM architecture of plxnd1 mutant VAT was markedlydifferent from sibling VAT (FIG. 3A), and characterized by larger andmore numerous interstitial collagen fibers (FIG. 3A, FIG. 11). Moreover,plxnd1 mutant VAT had increased glycoprotein, Elastin content (FIG. 12C)and a greater abundance of fibrous ECM (FIG. 12). plxnd1 mutant SAT didnot have altered collagen or fibrous ECM (FIG. 12D).

To assess fibrillogenesis in plxnd1 mutant VAT, ECM is extracted fromzebrafish VAT, and fibril polymerization and gel formation is induced invitro (FIG. 13). Turbidity assays are used to determine the rate andultimate extent of fibrillogenesis. ECM was purified from dissectedzebrafish VAT (˜100 VATS per tube, triplicate tubes per condition) aspreviously described. (Uriel S, et al. (2009), Tissue engineering. PartC, Methods 15 (3):309-321.) Urea extraction was allowed to proceed for14-21 days at 4° C. Protein concentration (˜7 mg/ml) was assessed by theBradford assay (Thermo Scientific). Protein concentrations were notsignificantly different between groups. To induce fibril polymerizationand gel formation, acetic acid was added to a pH of 7. Turbidity assayswere conducted as previously described using a Spectronic 20D+spectrometer (Thermo Scientific). (Wood G C & Keech M K (1960), TheBiochemical journal 75:588-598.)

Plxnd1-deficient VAT underwent an increased rate of fibrillogenesiscompared to sibling ECM (FIG. 3B), and plxnd1 mutant VAT attained ahigher ultimate turbidity than sibling VAT (FIG. 3C). Injection ofcol5a1-i1e2 vMO did not affect in vitro fibrillogenesis of sibling VAT;however, col5a1 vMO injection significantly reduced both the rate offibrillogenesis and turbidity in plxnd1 mutant VAT (FIGS. 3B & C).

plxnd1 mutant ECM is sufficient to induce hyperproliferation andhyperplastic morphology of SVCs in vitro

Isolated ECM from zebrafish VAT was used as a substrate for the cultureof primary stromal vascular cells (SVCs) also isolated from zebrafishVAT (FIG. 3D). Isolation of SVCs was conducted largely as describedpreviously (˜50 VATs per tube, triplicate tubes per condition) (BouraouiL, et al. (2008), The Journal of endocrinology 198 (3):459-469), exceptcollagenase incubation was undertaken at 28.5° C. and the cellsuspension was passed through a 70 μm cell strainer. Zebrafish SVCs werecounted using a hemocytometer and resuspended in actively polymerizingECM at 5×10⁵ cells/ml. Prior to addition of SVCs, purified zebrafish VATECM was diluted to 4 mg/ml. 100 μl of ECM gel and SVCs were used perwell of a 96-well plate. The 3D co-culture was maintained in DMEM growthmedia (Sigma, #D5796) containing 10% FBS, 2 mM L-glutamine, 10 mM HEPESas described (Bouraoui L, et al. (2008), The Journal of endocrinology198 (3):459-469) for 6 days at 28.5° C./5% CO2 with the addition ofPen/Strep (5K/5K) (Cambrex, #17-603E), 10 mg/ml Gentamycin (Sigma,#G1272) and 250 μg/ml Fungizone (Fisher, #BP928-250). Media was changeddaily. After 6 days, media was changed to differentiation media-growthmedia supplemented with 0.2 mg/ml human recombinant Insulin (Sigma,#I2643), 1 M 3-isobutyl-1-methylxanthine (Sigma, #I5879), 250 μMdexamethasone (Sigma, #D4902), 45 μg/ml cholesterol (Sigma, #C8667), 100μg/ml cod liver oil (Sigma, #C5650), 250 μg/ml polyoxyethylene sorbitanmonooleate (Sigma, #P8074) and 20 μg/ml D-α-tocopherol acetate (Sigma,#T3634). Cultures were maintained in differentiation media for 4 daysbefore fixing in 4% paraformaldehyde followed by brightfield or confocalimaging. Culture confluency was measured by thresholding brightfieldimages of whole wells based on pixel intensity, and LD sizes weremeasured as described above.

When cultured within a 3D ECM substrate obtained from sibling VAT,sibling SVCs were able to proliferate and readily differentiate intoadipocytes containing large LDs (FIG. 3F). By contrast, plxnd1 mutantSVCs cultured on ECM derived from plxnd1 mutant ECM reached a higherlevel of confluency (FIG. 3E, F and SI Appendix, FIG. S13A) togetherwith strikingly smaller LDs (FIG. 3G and FIG. 14A). The degree ofconfluency and LD hypertrophy were ECM extract dependent, as culturingsibling SVCs within ECM from plxnd1 mutants increased confluency andreduced LD size (FIGS. 3E & F). Conversely, culturing plxnd1 mutant SVCsin sibling ECM abrogated the hyperproliferation and smaller LD sizeobserved in the mutant experiment, leading to larger LDs morereminiscent of sibling:sibling co-cultures (FIGS. 3E & F). Moreover,combining ECM extract from siblings and plxnd1 mutants producedintermediate morphologies dependent on the proportion of wild-typesibling:plxnd1 mutant ECM (FIGS. 14B & C), suggesting that the capacityof plxnd1 mutant ECM to induce proliferation and hyperplastic morphologyis proportional to the amount of plxnd1 mutant ECM present. Injection ofcol5a1 vMO prior to ECM extraction abrogated the ability of plxnd1mutant ECM to induce proliferation and hyperplastic morphology incultured plxnd1 mutant SVCs (FIGS. 14D & F). These data demonstrate thatplxnd1 mutant ECM is sufficient to induce hyperproliferation of SVCs anda smaller overall size of LDs.

Example 4 Regional Lipid Deposition is Controlled by PLXND1

Lipid is preferentially deposited in SAT of plxnd1 mutants fed ahigh-fat diet

Daily immersion in 5% chicken egg yolk over the course of 2-3 weeks isused as a high-fat dietary supplement (HFD) to induce lipid accumulationand metabolism in zebrafish (Semova I., et al. (2012), Cell host &microbe 12 (3):277-288; Walters J W, et al. (2012), Chemistry & biology19 (7):913-925; Carten J D, et al. (2011), Developmental biology 360(2):276-285; Marza E, et al. (2005), Developmental dynamics 232(2):506-518.)

Zebrafish were subjected to daily exposures of 5% chicken egg yolk overthe course of 14-21 days. Wild-type Ekkwill, plxnd1 heterozygotes orplxnd1 homozygous mutant zebrafish were raised under normal conditionsuntil 30 days post-fertilization (dpf), then ˜10 size matched fish weretransferred to ‘nursery’ mesh bottom tubes (Aquatic Habitats, #RC33A)suspended within a regular 10 L tank on the main recirculating system.Use of nursery tubes allows the segregation of experimental groupswhilst maintaining identical environmental exposures and improved waterconditions afforded by a recirculating aquatic system. Whilst containedwithin the nursery tubes, fish were fed a normal diet (see above) withthe following supplement: on a daily basis, nursery tubes were moved totanks containing either 5% (% v/v) chicken egg yolk in system water(high-fat diet; HFD) (Latta's Egg Ranch, Hillsborough, NC) or systemwater (control diet). Nursery tubes were incubated in their respectivedietary regimen for ˜2-4 hours daily, before being rinsed with systemwater and placed back in the original 10 L tank where they continued toreceive normal diet. These daily supplements were continued for between14 and 21 days.

HFD treatment led to equivalent increases in lipid accumulation in bothplxnd1/+ heterozygotes and homozygous plxnd1 mutants (FIG. 4A, FIG.15A). A substantial increase in total VAT and SAT area (FIGS. 15B & C)and VAT and SAT LD size was observed in heterozygotes (FIGS. 4C & D).However, VAT in HFD fed plxnd1 mutants did not expand (FIG. 15B), andVAT-LDs did not substantially increase in size (FIGS. 4C & D). plxnd1mutants underwent a larger expansion of SAT when compared toheterozygotes (FIG. 15C), leading to supersized SAT-LDs (FIG. 4D) and andecreased VAT:SAT ratio (FIG. 4B). Together these data show that absenceof Plxnd1 results in a preferential expansion of SAT in response to HFD,and thus leads to further exacerbation of altered body fat distribution.

Plxnd1 deficiency protects zebrafish from high-fat diet induced insulinresistance

Glucose assessment and glucose tolerance test (GTT). Adult zebrafishwere weighed as described. (Eames S C, et al. (2010), Zebrafish 7(2):205-213.) 1 mg/g glucose (fish weight) was injectedintra-abdominally. 5 μl of blood was collected by cardiac puncture, andglucose levels assessed using the FreeStyle Lite monitor (Abbot DiabetesCare Inc) as described. (Eames S C, et al. (2010), Zebrafish 7(2):205-213.) For basal blood glucose assessments, zebrafish were fastedfor 4 h prior to blood collection.

HFD-fed wild-type siblings had hyperglycaemia (FIG. 5A). Further, aftera glucose tolerance test (GTT) siblings fed a HFD failed to normalizeblood glucose levels, suggesting a degree of systemic insulin resistance(FIG. 5B). By contrast, plxnd1 mutants fed a HFD did not exhibithyperglycaemia (FIG. 5A) and efficiently normalized hyperglycemia aftera GTT (FIG. 5B). The insulin receptor substrates 1 and 2 (irs1, irs2)are negatively regulated in insulin resistance. (Capiotti K M, et al.(2014), Comparative biochemistry and physiology. Part B, Biochemistry &molecular biology 171:58-65; Goodyear L J, et al. (1995), The Journal ofclinical investigation 95 (5):2195-2204; Ruiz-Alcaraz AJ, et al. (2005),The Biochemical journal 392 (Pt 2):345-352.) However, in both controland HFD fed plxnd1 VAT, irs1 and irs2 mRNAs were increased compared tosiblings supporting augmented insulin signaling in plxnd1 mutants (FIG.5C).

Example 5 Increased PLXND1 mRNA in Human VAT is Associated with Type 2Diabetes

Correlation between PLXND1 expression and adipocyte insulin sensitivity(FIG. 16) was examined in another cohort of 56 individuals comprising 30obese (BMI>30 kg/m2) otherwise healthy and 26 non-obese (BMI<30 kg/m2)healthy women. (Arner E, et al. (2012), Diabetes 61 (8):1986-1993.) Allwere pre-menopausal and free of continuous medication. An abdominal SATbiopsy was obtained by needle aspiration. Adipocyte in vitro insulinsensitivity was determined by quantifying the uptake of glucose intolipids in response to insulin as previously described. (Dahlman I, etal. (2004) alpha2-Heremans-Schmid glycoprotein gene polymorphisms areassociated with adipocyte insulin action. Diabetologia 47(11):1974-1979.) Microarray analysis was performed exactly as describedon fractionated abdominal SAT adipocytes (Amer E, et al. (2012),Diabetes 61 (8):1986-1993) using the Affymetrix GeneChip mRNA Arrayprotocol. Gene expression results are accessible at GEO (accessionnumber GSE25402). Multiple regression analysis was performed to assesscorrelation of PLXND1 and insulin sensitivity, adjusting for age andBMI.

PLXND1 mRNA levels in VAT and SAT from lean (N=8), healthy obese (N=8),and type 2 diabetic obese (T2D, N=8) patients were quantified bymicroarray (FIGS. 5D and 16C) exactly as described. (Dahlman I, et al.(2006), Diabetes 55 (6):1792-1799.) Groups were compared by Student'st-test. Values are mean±SD.

PLXND1 mRNA was specifically increased in VAT of obese patients withtype 2 diabetes (FIG. 5D). No change was observed in SAT (FIG. 5D) or inAT of healthy obese patients (FIG. 5D).

1. A method of treating metabolic dysfunction in a subject comprisingadministering to the subject a therapeutically effective amount of amolecule, wherein said molecule is capable of modulating anintracellular pathway mediated by PLXND1.
 2. The method of claim 1,wherein the molecule is a small molecule chemical compound, a nucleicacid molecule, a peptide, or a polypeptide.
 3. (canceled)
 4. (canceled)5. The method of claim 1, wherein the molecule directly modulates any ofthe genes, or gene products, for PLXND1, COL5A1, COL1A2, COL2A1, COL4A1,COL5A2, COL5A3, COL6A1, FN1, ACAN, LAMA1, GPC4, or SPARC.
 6. The methodof claim 1, wherein the molecule is an interfering molecule comprisingan antibody, an antibody fragment, an antisense RNA, a cDNA, adominant-negative form of a molecule, a peptide, or a protein kinaseinhibitor.
 7. The method of claim 1, wherein the molecule is an agonist.8. The method of claim 1, wherein the molecule is an antagonist. 9.(canceled)
 10. The method of claim 1, wherein the metabolic dysfunctioncomprises diabetes, type 2 diabetes, or insulin resistant diabetes. 11.The method of claim 1, is a regulator of extracellular matrix proteins.12. (canceled)
 13. A method of identifying a subject at risk formetabolic dysfunction, the method comprising: (a) isolating a biosamplefrom a subject; (b) determining a level of one or more biomarkerspresent in the biosample; wherein said one or more biomarkers is:PlexinD1 protein or nucleic acid, COL5A 1 protein or nucleic acid,COL1A2, COL2A1, COL4A1, COL5A2, COL5A3, COL6A1, FN1, ACAN, LAMA1, GPC4,or SPARC protein or nucleic acid visceral adipose tissue, subcutaneousadipose tissue, hypertrophic visceral adipose tissue, or hyperplasticvisceral adipose tissue; and (c) identifying the subject as having arisk for metabolic dysfunction when the concentration of one or morebiomarkers is increased or decreased relative to a control level orrange.
 14. The method of claim 13, wherein the one or more biomarkerscomprises at least two biomarkers.
 15. The method of claim 13, whereinthe one or more biomarkers comprises a ratio of biomarkers.
 16. Themethod of claim 15, wherein the ratio of biomarkers is comprised of alevel of visceral adipose tissue and a level of subcutaneous adiposetissue.
 17. The method of claim 13, wherein the metabolic dysfunctioncomprises a diabetic condition.
 18. The method of claim 17, wherein thediabetic condition comprises metabolic syndrome or type 2 diabetes. 19.(canceled)
 20. The method of claim 13, wherein the metabolic dysfunctioncomprises insulin resistance.
 21. (canceled)
 22. (canceled)
 23. Themethod of claim 13, wherein the biosample is comprised of biopsymaterial, adipose tissue, bone marrow samples, blood, blood plasma,serum or cellular fraction thereof, urine, feces, saliva, tears, orcells derived from a biological source.
 24. The method of claim 13,wherein determining the level of one or more biomarkers comprises PCR,RT-PCR, ELISA, immunolabeling, in situ hybridization, or nucleic acidsequencing.
 25. A method for identifying a subject that is eligible forreimbursement of an insurance claim for treatment of metabolicdysfunction, the method comprising: (a) isolating a biosample from asubject; (b) determining a level of one or more biomarkers present inthe biosample; wherein said one or more biomarkers is selected from:PlexinD1 protein or nucleic acid, COL5A1 protein or nucleic acid,COL1A2, COL2A1, COL4A1, COL5A2, COL5A3, COL6A1, FN1, ACAN, LAMA1, GPC4,or SPARC protein or nucleic acid visceral adipose tissue, subcutaneousadipose tissue, hypertrophic visceral adipose tissue, or hyperplasticvisceral adipose tissue; and (c) identifying the subject as eligible forreimbursement of the insurance claim when the concentration of one ormore biomarkers is increased or decreased relative to an insurancecontrol value.
 26. A method for determining the efficacy of a treatmentfor metabolic dysfunction in a subject, the method comprising: (a)treating a subject for a metabolic dysfunction; (b) isolating abiosample from the subject; (c) determining a level of one or morebiomarkers present in the biosample; wherein said one or more biomarkersis selected from: PlexinD1 protein or nucleic acid, COL5A 1 protein ornucleic acid, COL1A2, COL2A1, COL4A1, COL5A2, COL5A3, COL6A1, FN1, ACAN,LAMA1, GPC4, or SPARC protein or nucleic acid visceral adipose tissue,subcutaneous adipose tissue, hypertrophic visceral adipose tissue, orhyperplastic visceral adipose tissue; and (d) determining the efficacyof the treatment for metabolic dysfunction when the concentration of oneor more biomarkers is increased or decreased relative to a referencelevel of the one or more biomarkers.
 27. The method of claim 26, whereinthe reference level of one or more biomarkers is derived from apopulation of healthy subjects.
 28. The method of claim 26, wherein thereference level of one or more biomarkers is derived from a biosampleisolated from the subject prior to treating the subject for a metabolicdysfunction.